The goals of the research proposed are first, to characterize the effect of plasma on factor VIII binding to von Willebrand factor (vWF) and identify a vWF-binding protein that inhibits factor VIII binding. Aspects such as affinity and stoichiometry for the interaction of factor VIII with pure vWF vs. vWF reconstituted in plasma will be studied. It will also be determined whether the putative plasma vWF-binding protein decreases the rate of factor VIII association with vWF or increases the rate of dissociation from vWF. The importance of these findings relates to a better understanding of modulation of factor VIII levels in pregnancy, inflammation and DDAVP therapy, as well as secondary factor VIII deficiency. Until now, the explanation of secondary factor VIII deficiency is limited to a "decreased binding" of factor VIII to vWF due to specific mutations in the factor VIII binding region of vWF. Understanding binding kinetics of the association of another protein with vWF will help to better characterize secondary factor VIII deficiency. The second goal of this proposal is to identify structural features of vWF that influence the interaction with factor VIII. To accomplish this aim, we will focus on studies to characterize the role of inter and intrasubunit vWF disulfide bonds on factor VIII binding. We will conduct equilibrium and kinetics studies that will compare binding of factor VIII to multimeric vWF and mutant vWF incapable of multimer formation. In addition, we will construct vWF mutants based on mutations found in a patient with secondary factor VIII deficiency (R9lQ and C268S), and investigate what is the role of vWF Cys 268 on factor VIII binding. vWF regulates the levels of factor VIII, which has been shown to increase its plasma levels in deep venous thrombosis (DVT). Our results may alter the understanding of pathogenesis of DVT.